Translational inhibition by 5′-polycytidine tracts in Xenopus embryos and in vitro

• Published in: Gene 1996-10, Vol.176 (1), p.17-21
• Main Authors: Kuo, John S., Veale, Robin, Maxwell, Bridey, Sive, Hazel
• Format: Article
• Language: English
• Subjects: 5'-Leader sequences; Animals; Base Sequence; cDNA library; Cytidine; Molecular Sequence Data; mRNA expression; Poly C; Protein Biosynthesis; Recombinant DNA; RNA, Messenger; SP6 RNA polymerase; T7 RNA polymerase; Xenopus laevis; Xenopus laevis - embryology; Xenopus laevis - genetics
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(96)00201-6

Introduction of in vitro transcribed mRNA into Xenopus laevis embryos is a useful technique for analyzing gene function. In order to optimize expression of cDNA constructs from in vitro transcribed mRNAs, we examined the translational efficiency of reporter genes that simulated cDNAs synthesized by tailing with deoxyguanosine (dG) before second strand cDNA synthesis. When transcribed in vitro, these cDNAs give rise to RNAs containing a 5'-homopolymeric cytidine (poly(C)) stretch. We observed that the presence of a 5'-poly(C) tract depressed translation of a CAT reporter gene at least 100-fold in Xenopus embryos and up to 5-fold in vitro. This effect was not seen when a 5'-polyadenosine tract was tested. Translational depression was dependent on the phage polymerase used for in vitro transcription: RNAs transcribed by T7 polymerase translated far more poorly than those transcribed by SP6 polymerase. These results have general implications for optimizing expression of cDNA constructs.